SOLUTION X-RAY SCATTERING STUDIES ON ALLOSTERIC TRANSITION OF E COLI ASPARTATE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 大肠杆菌天冬氨酸氨基转移酶是一种典型的变构酶，具有同向性和异构性的协同作用，在嘧啶生物合成过程中起着反馈调节的作用。我们结合了x射线结晶学和溶液x射线溶液技术来研究与变构转变相关的大尺度的四元结构变化。由结晶学获得的高分辨结构有时会受到晶胞物理约束的影响，而溶液散射被用来评估晶体结构以及在溶液中获得真实的生理结构。溶液X射线散射常被用来研究配体结合后结构变化的时间过程。我们最近研究了ATCase的几个突变版本，通过平衡和时间分辨溶液X射线散射，所有突变版本的ATCase都包含活性位点中关键氨基酸残基的取代。两个突变酶H134A和R167Q在加入饱和浓度的双底物类似物N-膦-乙酰基-L-天冬氨酸后保持T状态，而另外两个突变酶R229A和Q231L仅部分向R状态移动。在加入天然底物(S52A、K84A、Q231L和R296A)后，确实表现出向R状态转变的突变酶的T-&GT；R转换速率都比野生型酶慢得多。最引人注目的是，加入PALA后，这些突变酶的T-GT；R转换率与野生型酶相比没有变化。这些数据表明，失去结合位点上的单一相互作用并不足以消除酶经历T到R转变的能力，除非进行这种相互作用的残基也参与了结构域间R状态稳定的盐键或与其他活性位点残基形成氢键。此外，这些数据表明，与天然底物相比，PALA结合后的变构转变途径不同

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. E. coli aspartate transcarbamoylase is a classical example of allosteric enzyme, possessing homotropic and heterotropic cooperativities, which have a fundamental role in feedback regulation in the pyrimidine biosynthesis pathway. We combine x-ray crystallography and solution x-ray solution techniques to study a large scale quaternary structure change associated with the allosteric transition. High resolution structures obtained by crystallography are sometimes biased by the physical constraints of the unit cell, and solution scattering is used to evaluate crystal structures as well as to obtain true physiological structures in solution. Solution x-ray scattering is frequently used to investigate time-course of structural change upon ligand binding. We have recently investigated several mutant versions of ATCase, all containing substitutions of critical amino acid residues within the active site by equilibrium and time-resolved solution x-ray scattering. Two mutant enzymes H134A and R167Q, remained in the T state after addition of a saturating concentration of the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA), and two others, R229A and Q231L, were shifted only partly towards the R state. The mutant enzymes that did exhibit a shift towards the R state after addition of the natural substrates (S52A, K84A, Q231L and R296A) all had much slower T -> R transition rates than the wild-type enzyme. Most strikingly the T -> R transition rate for these mutant enzymes after addition of PALA was unchanged as compared to the wild-type enzyme. These data indicate that the loss of a single interaction in the binding site is not enough to eliminate the ability of the enzyme to undergo the T to R transition, unless the residue making that interaction is also involved in an interdomain R-state stabilizing salt link or forms hydrogen bonds with other active site residues. In addition, the data suggest different pathways for the allosteric transition after the binding of PALA as compared to the natural substrate